SUMMARY - Human GBPs as regulators of immunity to intracellular bacterial pathogens The guanylate binding protein (GBP) family of host defense proteins has been shown to play diverse and critical roles in cell-autonomous immunity to intracellular bacterial pathogens. These proteins are major regulators of fundamental host defense modules and involved in processes such as inflammasome activation and the execution of direct bactericidal activities. However, almost all of this work has been performed in mouse models and antimicrobial functions of human GBPs (hGBPs) are still poorly characterized. In our recent studies we identified divergent host defense functions of the human GBPs and linked specific human GBPs to novel resistance mechanisms that protect against an important class of bacterial pathogens. Specifically, we found that human GBP1 (hGBP1) underpins two novel host defense mechanisms against cytosolic Gram-negative bacterial pathogens: 1) hGBP1 protein directly interferes with pathogen-induced actin-based motility, resulting in pathogen restriction; and 2) hGBP1 mediates activation of chemokine production by infected epithelial cells. Thus, hGBP1 activation appears to serve as a central node for two distinct but synergistic immune functions: a cell-intrinsic defense program prevents cell-to-cell spread of the pathogen while the production of paracrine immune signals promotes the activation and recruitment of immune cells to the site of infection. In this work, we will probe mechanistically these two important roles for hGBP1 that we have uncovered. In Aim1 we will combine cell culture and biochemical approaches to define the mechanism by which hGBP1 blocks actin-based motility for the cytosolic bacterial pathogens Shigella flexneri and Burkholderia thaliandensis. In Aim2 we will explore a second, independent function of hGBP1 as a novel regulator of an immune sensing pathway leading to the production of immune-modulatory chemokines. We also discovered that the human-adapted pathogen Shigella flexneri but not the nonpathogenic bacillus Burkholderia thaliandensis evolved strategies to block hGBP1-mediated immunity. Accordingly, we will dissect the mechanism by which Shigella reverses hGBP1- mediated inhibition of actin-based motility in Aim3 using bacterial genetics, host genetics and cell biological approaches. Overall, the work proposed here will provide a fundamental understanding of the role of hGBP1 and other human GBPs in cell-autonomous immunity to intracellular bacterial pathogens. Deciphering the interplay between GBP-mediated immunity and bacterial counter-defense could provide a path towards novel antimicrobial interventions. !